Significant improvements in the treatment of lung diseases (e.g. asthma, COPD, cystic fibrosis (CF)) have occurred over the past 30 years due to drug delivery via inhalation aerosols. However, the efficacy inhaled therapies is dramatically reduced in these patients because of the presence of a viscous mucus transport barrier within the airways, extensive degradation and metabolism of inhaled drug prior to exerting its pharmacological action, and the development of other barriers within the airways due to infection and inflammation, such as in the case of biofilms produced by mucoid Pseudomonas aeruginosa colonies. Often drugs or gene vectors cannot reach the intended target before their activity has been reduced or eliminated. Poor transport efficiencies in drug delivery have lead to the failure of therapies including the unattainability of the relatively low efficiencies required for gene therapy success.
Cystic fibrosis is an excellent example of a lung disease in which extracellular barriers prevent effective drug delivery to the lungs. Understanding the genetic defect underlying CF has been described as only half the battle in finding the cure for this disease. This has been evidenced in recent years when, despite great hopes, gene therapy cures have failed to materialize. The underlying cause of CF is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This protein acts as a chloride ion channel and is found in exocrine glands and secretory epithelia. The defective CFTR protein in the respiratory tract reduces the secretion of chloride ions and alters the nature of respiratory mucus. This leads to respiratory complications that include airway obstruction, chronic lung infection, and inflammatory reactions. In nearly all cases, complications in the lungs determine the quality of life/life expectancy of patients.
Pseudomonas aeruginosa as the most common bacterial pathogen causing infection in the lungs of people with CF and appropriate antibiotic therapy is vital. Lower respiratory tract infection with Pseudomonas aeruginosa (P. aeruginosa) occurs in most people with cystic fibrosis. Once chronic infection is established, P. aeruginosa is virtually impossible to eradicate and is associated with increased mortality and morbidity’. Antibiotics for exacerbations are usually given IV, and for long-term treatment, via a nebulizer as an aerosol.
Current treatment options include targeted therapy using inhalation aerosols and have been reported since the 1940's. Now, as with asthma, aerosol drug administration in CF appears to have matured and is often used first in the treatment of opportunistic bacterial infections. Inhaled antibiotics have several significant advantages over IV therapy as a large pharmacokinetic advantage is afforded by directly delivering the drug to the airway lumen, avoiding a systemic exposure and potential toxicity. Moreover, inhaled antibiotic therapy administered in the patients' home is significantly cheaper than IV therapy either at home or in the clinic. For P. aeruginosa, several clinical trials have compared a nebulized anti-pseudomonal antibiotic with placebo or usual treatment (oral or IV antibiotics). Lung function was better in the treated group than in a control group. However, resistance to antibiotics increased more in the aerosolized antibiotic treated group than in the placebo. Although outcomes generally favor the aerosol delivery of anti-pseudomonas therapies, it is apparent that drug resistance, through mechanisms including chronic sub-therapeutic concentrations (poor penetration) and mucoid P. aeruginosa development, may be responsible.
Another treatment option is gene therapy. By far, gene therapy has been researched more in CF than any other disease due to the feasibility of theoretically correcting the disease by providing a single copy of the CFTR gene to airway epithelial cells. Clinical trials in patients with CF have provided proof-of-principle for gene transfer to the airway epithelium; however, gene delivery is inefficient. Most of the many clinical trials initiated have now been discontinued owing to unsatisfactory primary outcomes. Several studies have shown that extracellular barriers such as mucus and sputum and mucociliary clearance significantly reduce transfection efficiency.
Biophysical barriers to be overcome in treatment of CF are now discussed.
1) Barriers of Aerosol Delivery for CF:
Efficient drug delivery is significantly limited by the multitude of biological and physical barriers in CF. The primary barrier is the viscous mucus layer that results from abnormal secretions. As a consequence of the increase in viscosity/elasticity of the CF mucus, the mucociliary clearance mechanism for removing inhaled particles and microbes is dramatically reduced. This breakdown of the mucociliary escalator results in colonization of the lung and the development of an inflammatory response that imposes additional barriers to successful therapeutic treatment of the airway surface. These include: (1) an extensive inflammatory milieu that causes oxidative or enzymatic degradation of inhaled therapeutics, (2) release of bacterial and endogenous cellular breakdown products and contents (most significantly DNA and lipids), (3) development of mucoid P. aeruginosa infections that produce and secrete the exopolysaccharide alginate, and (4) airway obstruction.
2) The Mucus Barrier:
It is well known that the mucus blanket in CF protects the epithelial cells by forming a diffusion barrier for inhaled particles. Respiratory mucus is a complex material, which possesses both flow and deformation rheological properties, characterized by non-linear and time-dependent viscoelasticity and physical properties of adhesiveness and wettability. Viscosity and elasticity are directly involved in the transport capacity of mucus, whereas wettability and adhesiveness contribute to the optimal interface properties between the mucus and the epithelial surface 18. Different biochemical constituents, such as glycoproteins, proteins, proteoglycans, and lipids are involved in the gel properties of respiratory mucus. During bronchial infection and particularly in CF, the loss of water and the increase in macromolecules result in a marked increase in viscosity and adhesiveness responsible for the mucus transport impairment. Phospholipids and associated mucins are also implicated in the interaction between bacteria and epithelial cells.
3) Drug Transport Through Mucus Barriers:
It has been observed that gastrointestinal mucus retards the diffusion of macromolecules. Lipids are a major contributor to reduced diffusion of drugs in native intestinal mucus. In gene therapy, transfection is markedly inhibited in the presence of sputum. Removal of sputum before gene transfer showed increases in efficiency. Pretreatment of sputum-covered cells with DNase also improved gene transfer but efficiencies remained low. It has also been noted that in vivo transfection appears to be a thousand times less efficient than in vitro transfection. This has been attributed to differences in cell characteristics and noncellular barriers. In vivo, the target cells for gene therapy in CF are the submucosal gland cells and the epithelial cells lining the small conducting airways in the lungs. Thus, gene transfection systems must permeate the overlying mucus layer in order to be effective. Compared with normal airway secretions, CF mucus has a higher visco-elasticity because of a high content of actin, serum proteins, DNA, alginate, and rigidifying lipids. These negatively charged biopolymers (mucin, DNA, and alginate) are connected to each other through physical entanglements of their chains and noncovalent interactions. The result is a viscoelastic network that prevents drugs and genes from efficiently being distributed and transported to their target sites.
4) Degradation and Inflammatory Effects:
Neutrophils account for 1% of the inflammatory cell population in epithelial lining fluid in normal individuals, but in CF, neutrophils constitute 70% of the inflammatory cells. Because the neutrophil is remarkably active in the production of degradation enzymes and associated species, the local environment of the CF airways is very inhospitable for therapeutic agents.
5) Barriers Presented by Bacterial Colonization of the Lung:
Although multiple microbial species can successfully colonize the CF lung, robust infections by P. aeruginosa eventually dominate the microbial population and become the major contributor to disease severity and life expectancy. The conversion of P. aeruginosa microcolonies from a non-mucoid to a mucoid phenotype marks the transition to a more persistent state, characterized by antibiotic resistance and accelerated pulmonary decline. The known and proposed roles of alginate in mucoid infections includes (1) generation of an alginate capsule for direct barrier to phagocytosis and opsonization, (2) immunomodulatory effects, and (3) biofilm related phenomena such as bacterial adhesion and antibiotic resistance. The interference of the exopolysaccharide barrier with antibiotic penetration was demonstrated. Penetration of positively charged hydrophilic drugs, such as aminoglycosides and polypeptides, was markedly inhibited. Even the most aggressive antibiotic treatments may have limited effect on mucoid colonies because of this barrier. Moreover, even sensitive bacteria that do not have a known genetic basis for resistance can have profoundly reduced susceptibility when they form a biofilm. When bacteria are dispersed from a biofilm, they usually rapidly become susceptible to antibiotics, which suggests that resistance of bacteria in biofilms is not acquired via mutations or mobile genetic elements.
6) Airway Obstruction:
Due to chronic infection, mucus accumulation, and airway remodeling, the airways of CF patients can become acutely or chronically obstructed. This can pose significant problems for aerosol therapies that utilize larger aerodynamic particle sizes. Many inhaled antibiotics, for example, are administered using the same design of nebulizer as those used in asthma. As in the case of serious acute asthma attacks, the delivery of aerosols in severely obstructed airways will result in significant decreases in therapeutic doses to the lungs.
Up to now, magnetic nanoparticles have been used in medicine for magnetic separation techniques, as contrast agents in magnetic resonance imaging, for local hyperthermia, or as magnetic targetable carriers for several drug delivery systems. These kinds of particles are routinely produced as commercial contrast agents for MRJ investigations (Combidex®, Resovist®, Endorem®, Sinerem®). These iron oxide nanoparticles exhibit superparamagnetic properties and can be attracted by an external magnetic field. As a result, several in vitro and in vivo studies have shown this to be a promising strategy of localizing chemotherapy agents at cancer tumor sites for cancer therapy.
Similar to magnetic nanoparticles, thermally active nanosystems have been reported for the treatment of cancer via thermal ablation of tumor cells. Several types of metal nanoparticles are capable of converting energy, such as that carried by near-infrared light or an oscillating magnetic field, into heat at a level high enough to kill tumors; i.e. by inducing localized heating in an in vivo situation. Recently, a Phase I clinical trial was completed in patients with recurrent prostate cancer. This study showed that magnetic nanoparticles could be safely administered to humans and produced localized tumor-killing temperatures when stimulated by an oscillating magnetic field. The investigators injected biocompatible magnetic iron oxide nanoparticles directly into the patients' tumors using ultrasound and fluoroscopic imaging to guide the injections. Then, using a magnetic field applicator designed specifically for administering thermal anticancer therapy, the nanoparticles were excited. Each treatment lasted 1 hour and was repeated weekly for 6 weeks. Intracellular hyperthermic treatment of tumors differs from the thermal ablation described above. Intracellular heating has been shown to be incapable of heating the solution or general tumor environment. However, due to the apparent very high temperatures well localized within the cell, cytolysis was induced when an external alternating magnetic field was applied with the biological barriers in CF and how the thermal nanoparticles will interact with the therapeutic agents. It is possible that these systems could be tethered together or be released separately.
As discussed above, CF is characterized by the presence of major barriers to drug and gene delivery. Pre-clinical and clinical studies of gene transfer for CF are ongoing, but these have demonstrated a poor ability to achieve efficient gene transfer through CF mucus. For both gene and drug delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. An object of the present invention is to provide active multifunctional nanoparticles and methods for enabling enhanced drug delivery and therapeutic efficacy by overcoming these biological barriers in CF as well as biophysical barriers in the delivery of therapeutics to the airways of patients with cystic fibrosis and infectious lung disease.